You will be asked about differential staining, gram positive and. Microbiology lab exam 1 from turned in labs flashcards. Gram staining is a method commonly used to determine the chemical make up of the cell wall of bacteria. Flood the smear with gram s iodine solution and leave for 1 minute. The gram stain, the most widely used staining procedure in bacteriology. Gram stain principle, reagents, procedure, steps, results. A gram stain is a laboratory procedure used to detect the presence of bacteria and sometimes fungi in a sample taken from the site of a. It is a primary test which quickly and cost effectively divides bacteria into one of two types.
An infected appendix, or any tissue containing both negative and positive gram rods. If the slide is heat fixed, allow it to cool to the touch before applying the stain. Streptococcus pneumoniae is gram stain positive see more. A positive gram stain result means the bacteria has a thick peptidoglycan layer in the cell wall that retains the stain after it is washed away from the rest of the sample. Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent. Read more gram stain principle, reagents, procedure, steps, results. Method of staining used to distinguish and classify bacterial species into two large groups.
The gram stain procedure, developed by hans christian gram in 1884, relies on the differential cell wall staining properties of gram positive and negative bacteria. Gently flood the smear with grams iodine and let stand for 1 minute. How to perform a gram stain mccc microbiology youtube. The procedure is based on the reaction between peptidoglycan in the cell walls of some bacteria. Gram staining is an empirical method of differentiating bacterial species into two large groups gram positive and gram negative based on the chemical and physical properties of their cell walls. Pdf gram staining is an empirical method of differentiating bacterial species into two large groups grampositive and. Would you be able to visualize free endospores using the gram stain. The gram staining technique differentiates the mixed culture cells into two terms as gram positive bacteria and gram. Describe the composition of a gram positive cell wall and indicate the possible beneficial functions to the bacterium of peptidoglycan, teichoic acids, and surface proteins. The gram stain is a classical and important staining technique that remains widely used by. Knowing the chemical make up makes it easier to manipulate the bacteria for various purposes. The cell wall can stain either positive or negative, depending on its chemistry. Flood the gram s iodine for 1 minute and wash with water.
Through a series of staining and decolorization steps, organisms in the domain bacteria are differentiated according to cell wall composition. Gram stain developed in 1884 by hans gram, a danish microbiologist. Procedure of endospore stain spore staining procedure. Microfiltration has become a popular procedure for the concentration and enumeration of bacteria. Gram staining of bacteria from environmental sources.
Notes on the gram stain with description of a new method. Please explain in some detail why gram positive cells stain purple using the gram stain, while gram negative cells stain pink red. What are the factors that affect the gram staining procedure. Which reagent serves this purpose mordant in the gram stain procedure. Gram staining 2 free download as powerpoint presentation. Place slide with heat fixed smear on staining tray. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. Procedure, results and uses learn microbiology online medium used for urease test. Grams stain is a laboratory staining technique that distinguishes between two.
Gram positive bacteria gram negative bacteria provides useful information for disease treatment. Place slides on staining rack, drop crystal violet stain onto tissue section, stain for 1 minute. Laboratory perspective of gram staining and its significance in. This video demonstrates the gram staining method for identifying. Gramnegative cells have a thinner peptidoglycan layer that allows the crystal violet to wash out. Free microbiology photos of gram stained slides viewed through compound microscope and with naked eye. Gram staining stems cell effects grampositive gramnegative. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then blot dry with absorbent paper. It allows a large proportion of clinically important bacteria to be classified as either gram positive or negative based on their.
The gram stain, the most widely used staining procedure in bacteriology, is a complex and differential staining procedure. There are various factors that can affect the gram staining procedure such as the age of culture or type of growth medium. Rapid method for detection of grampositive and negative. Please note that the quality of the smear too heavy or too light cell concentration. Of all the different classification systems, the gram stain has withstood the test of time. The relationship between the nature of the cell wall and. Gram in 1884 it remains an important and useful technique to this day.
The name comes from the danish bacteriologist hans christian gram, who developed the technique. Walls of gram positive bacteria are richer in mucopeptide than are those of gram negative bacteria. Gram staining is a simple procedure if done with precision and accuracy can guide in diagnosis of several bacterial infections. In spite of his defective method, his name lives on jay hardy, cls, sm ascp hile working with infected lung tissue. Besides the picture is good, but a photo showing gram positive an d negative bacteria side by side would be much better.
I think the procedure how gram staining is done is important enought to be mentioned in wikipedia. The most widely used staining procedure in microbiology is the gram stain, discovered by the danish scientist and physician hans christian joachim gram in 1884. Any urea medium, agar or broth it can be a sole medium or part of panel like motility indole. None of the classes of major chemical constituents of the walls of bacteria and yeasts is exclusive to organisms which give a gram positive reaction. The latter are characterized by high lipid contents. The gram stain involves staining bacteria, fixing the color with a mordant, decolorizing the cells, and applying a counterstain. Microbiology lecture 2 gram positive and gram negative. This was developed in 1884 by a danish bacteriologist, hans christian gram. The gram stain easily divides bacteria into two groups, grampositive and gramnegative, on the basis of. Effect on reaction to the gram stain in staphylococcus aureus. The objective of gram stain this test differentiates the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiation of microorganisms.
Prepare the smear of suspension on the clean slide with a loopful of sample. Gram staining is a differential staining technique that differentiates bacteria into two groups. Grampositive cells have a thick layer of peptidoglycan in the cell wall that retains the primary stain, crystal violet. Differential stains render one type of microbe one color and other types of microbes another color. How to do a gram s stain test gram s stain is one of the most frequently used processes in identifying bacteria and is used daily in hospitals. However, the major factor that can influence the grain staining procedure is their different cell walls. Drain off the iodine wash the slide for the again in a gentle stream of tap water. Pdf gram staining procedure find, read and cite all the research you need on researchgate. Gently flood smear with crystal violet and let stand for 1 minute. This step does not remove the crystalviolet stain trapped within the gram positive cell wall. The most useful staining procedure in medical microbiology. The microscopicanalysis using gram staining technique showed that majority of the bacterial isolates were gram positive bacteria while only three of the isolates were gram negative bacteria. State what color gram positive bacteria stain after the gram stain procedure. The relation of the gram stain to the cell wall and the ribonucleic acid content of the cell.
Gram staining differentiates bacteria by the chemical and physical properties of their cell walls. Describe the function of heating the smear after application of the primary stain in the acid fast stain procedure. Occurrence of a phosphoric ester in certain bacteria. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet. Observe the results of the staining procedure under oil immersion using a brightfield microscope. Crystal violet was poured and kept for about 30 seconds to 1 minutes and rinse with water. List the reagents of the gram stain technique in order and their general rolee in the staining process. Please note that the quality of the smear too heavy or too light cell concentration will affect the gram stain results.
Old cultures of gram positive cells may not retain stain as well as younger cultures and could give false negative results ex pink cells. Gram staining technique is the widely used differential staining technique in bacteriology. Mccc bio201 microbiology laboratory instructional videos. History of gram stain the gram stain procedure was developed in 1883 by hans christian gram, a danish physician, who was working as a pathologist on a method he hoped would. The gram stain was developed by christian gram in 1884 and modified by hucker in 1921. Prepare smears of organisms to be tested for the presence of endospores on a clean microscope slide and air dry it. A page of biology 101 summarizing some species of pathogenic bacteria with a free pdf study guide.
Distinguishes bacteria of two large and medically important groups. This test differentiates the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiation of microorganisms. Cover the smear with crystal violet stain and leave for 1 minute. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
We developed a rapid and sensitive method for the differentiation of gram positive and gram negative bacteria, utilizing a polycarbonate membrane filter, crystal violet, iodine, 95% ethanol, and 6% carbol fuchsin, that can be completed in 60 to 90 s. Student procedures illinois institute of technology. In the gram stain, an insoluble crystal violetiodine complex is formed inside the cell, and this complex is extracted by alcohol from gramnegative but not from grampositive bacteria. Microbiology lecture 2 gram positive and gram negative bacteria gram staining principle this second microbiology lecture will explain the properties of gram positive and gram. Get a printable copy pdf file of the complete article 3. These online assessment tools will help you strengthen your understanding of the gram stain method. In the gram stain, gram positive organisms figure 3. Mature, free endospores should not be associated with the vegetative bacteria and should be seen as green ellipses. Rapid method for the differentiation of grampositive and.